No abstract

Steady-state and time-resolved fluorescence spectroscopy have been used for the study of the incorporation kinetics of hypericin (Hyp) into low-density lipoproteins (LDL). Biphasic kinetics of Hyp association with LDL was observed when solutions of Hyp and LDL were mixed at various concentration ratios. The rapid phase of Hyp incorporation is completed within seconds, while the slow phase lasts several minutes. The relative contributions of the individual phases show that a higher amount of Hyp molecules (65%) are incorporated into LDL in the second phase. The kinetics of the incorporation of Hyp into LDL particles preloaded with Hyp (Hyp/LDL=25:1) was also investigated. The decreased intensity of Hyp fluorescence is a sign of the formation of Hyp aggregates after penetration of additional Hyp molecules into Hyp/LDL=25:1 complex. The time dependence of Hyp fluorescence was measured after mixing the complex Hyp/LDL =200:1 with appropriate amounts of free LDL molecules. For each final Hyp/LDL ratio, an increase in the intensity and lifetime of Hyp fluorescence was observed, suggesting a monomerization of Hyp aggregates. The half-time of Hyp transfer from Hyp/LDL complex to LDL particles is similar to the half-time of the slow phase of Hyp incorporation into free LDL particles.

The aim of this study is to show that SR‐FTIRM can be successfully applied for the detection of early apoptotic processes in the human glioma cells (U‐87 MG). The apoptosis was induced by photodynamic action after irradiation of either photosensitizer hypericin (Hyp) or a complex of Hyp with low‐density lipoproteins (Hyp/LDL) incorporated into the cells. The differences between infrared (IR) spectra of non‐treated and photodynamically treated U‐87 MG cells are mainly manifested in position of Amide I vibrational band, sensitive to changes in protein secondary structure. The conformational transition α ‐helix to β ‐sheet results in the shift of Amide I vibration to lower frequency, and can be explained as a consequence of the processes leading to apoptosis. It is also shown that the sensitivity of SR‐FTIRM for the detection of early apoptotic signs is comparable, or even higher, than that of classic technique usually used for the detection of early apoptosis, flow‐cytometry study of cell staining by Annexin V. Moreover, we have demonstrated by both methods, SR‐FTIRM and flow‐cytometry, that Hyp/LDL complex loaded U‐87 MG cells 24 hours after photodynamic action are mostly late apoptotic/necrotic. In contrast, U‐87 MG cells loaded with Hyp alone display apoptosis as prevailing mode of cell death at 4 hours and 24 hours after photodynamic action. (© 2010 by Astro Ltd., Published exclusively by WILEY‐VCH Verlag GmbH & Co. KGaA)

The effect of alcohols on cell membrane proteins has originally been assumed to be mediated by their primary action on membrane lipid matrix. Many studies carried out later on both animal and yeast cells have revealed that ethanol and other alcohols inhibit the functions of various membrane channels, receptors and solute transport proteins, and a direct interaction of alcohols with these membrane proteins has been proposed. Using our fluorescence diS-C3 (3) diagnostic assay for multidrug-resistance pump inhibitors in a set of isogenic yeast Pdr5p and Snq2p mutants, we found that n-alcohols (from ethanol to hexanol) variously affect the activity of both pumps. Beginning with propanol, these alcohols have an inhibitory effect that increases with increasing length of the alcohol acyl chain. While ethanol does not exert any inhibitory effect at any of the concentration used (up to 3%), hexanol exerts a strong inhibition at 0.1%. The alcohol-induced inhibition of MDR pumps was detected even in cells whose membrane functional and structural integrity were not compromised. This supports a notion that the inhibitory action does not necessarily involve only changes in the lipid matrix of the membrane but may entail a direct interaction of the alcohols with the pump proteins.